Carcinogenesis is a multi-step process involving the activation of cellular proto-oncogenes and inactivation of tumor suppressor genes. Epithelial cells undergo different stages of phenotypic and genotypic alterations and gradually acquire malignant growth characteristics. The goals of our study are to develop an in vitro model of oral carcinogenesis using immortalized human gingival keratinocytes (IHGK) and to characterize the growth and differentiation properties of normal human gingival keratinocytes (NHGK), IHGK and a number of head and neck squamous cell carcinoma (HNSCC) lines and to relate the changes in phenotype with the gene expression profiles. We have conducted a systematic characterization of gene expression profiles in 25 HNSCC cell lines, and 1 IHOK transformed by HPV16 E6/E7 genes, using human cDNA microarrays containing 10K clones. NHOK were used as a reference. Our study shows that genes primarily involved in cell cycle regulation, oncogenesis, cell proliferation, differentiation, apoptosis and cell adhesion were widely altered in all 26 cell lines studied. Up-regulated genes included known oncogenes, protein kinases, DNA binding proteins and cell cycle regulators. While those commonly down-regulated included differentiation markers, cell adhesion proteins, extracellular matrix proteins, structural proteins (keratins) and protease inhibitor proteins. Compared to NHOK, a notable reduction in the expression of genes involved in terminal differentiation and cell adhesions was observed in HNSCC, suggesting that a loss in this process is an important signature of HNSCC. In addition, hierarchical clustering analysis of the data revealed two distinctive subtypes of gene expression patterns among the 26 cell lines, reflecting a degree of heterogeneity in HNSCC. By applying Significance Analysis of Microarrays, 136 genes were selected for being distinctively expressed between the two groups. Genes differentially expressed in the two subgroups include cell proliferation related genes, IGFBP6, EGFR, and VEGFC; tumor suppression and apoptosis related genes such as CASP4 (caspase 4), Tp53, Tp63; as well as cell cycle regulators such as CCND1 and CCND2 (cyclin D2) suggesting that the two subgroups might have undergone different pathways of carcinogenesis.